Figure 1.
β-arrestin2 recruitment to CXCR7 is dependent on C-terminal Ser/Thr residues.
(A) CXCL11 or CXCL12-mediated β-arrestin2 recruitment to CXCR7. HEK293T co-expressing RLuc-tagged CXCR7 and YFP-tagged β-arrestin2 were stimulated with increasing concentrations of CXCL11 (open circles) or CXCL12 (filled circles) (B) HEK293T co-expressing RLuc-tagged CXCR7 and YFP-tagged β-arrestin2 were incubated overnight with 25 ng/ml of PTX or for 30 min with the CXCR7-specific antibody 8F11 prior to the BRET measurement. (C) CXCL12-induced β-arrestin2 recruitment to CXCR7 wt (filled circles), a truncated CXCR7 lacking the C-terminus (CXCR7 ΔC, filled triangles) or a mutant CXCR7 for which all the Ser and Thr residues were mutated to Ala (CXCR7 ST/A, open squares). HEK293T cells coexpressing RLuc-tagged CXCR7 mutants and YFP-tagged β-arrestin2 were stimulated with increasing concentrations of CXCL12 prior to BRET measurements. Data represent the mean ± SEM of 4 experiments each performed in triplicate. Results are expressed in Net BRET as described in Materials and Methods. ***, p<0.001 by one-way ANOVA and Bonferroni post test.
Figure 2.
(A) CXCR7 internalization depends on CCPs and is G protein-independent.
HEK293T cells were transfected with wt CXCR7 (and β-arrestin (319–418) were indicated) and cell surface levels of the receptor after CXCL12 stimulation was detected by ELISA using the CXCR7-specific antibody 11G8. Incubation with 0.4 M Sucrose was done 30 min prior and during stimulation. PTX was incubated overnight at 25 ng/ml final concentration. (B) β-arrestin1/2 knock-down prevents CXCR7 internalization. HEK293/CXCR7 cells transfected with control siRNAs (white bars) or pools targeting β-arrestin1/2 (filled bars), were stimulated with CXCL12 (10−8 M) or vehicle for 45 min and receptor surface expression was determined. Knockdown of β-arrestin1 and -2, 48 hrs after transfection, was assessed in western blot using an anti-β–arrestin1/2 antibody (inset). Anti-STAT3 (mAb 79D7, Cell Signaling Technologies) was used as loading control. (C) CXCR7 C-terminus is essential for receptor internalization. HEK293T cells were transfected with wt CXCR7 (filled bars), CXCR7 ΔC (grey bars) or CXCR7 ST/A (white bars) and cell surface receptor levels were assessed as above. Data represent the mean ± SEM of at least 3 experiments each performed in triplicate. ***, p<0.001 by one-way ANOVA and Bonferroni post test.
Figure 3.
CXCR7 recycles to the cell surface after internalization.
(A) HEK293T stably expressing CXCR7 were stimulated with 10−8 M CXCL11, CXCL12 or vehicle for 45 min or 3 h and fixed immediately. CXCR7 was detected using the specific 11G8 antibody and an Alexa-488-conjugated secondary antibody. Scale bar represents 10 µm. (B) HEK293T cells expressing CXCR7 (filled symbols) or CXCR3 (open symbols) were incubated with CXCL11 (10−8 M, squares), CXCL12 (10−8 M, triangles) or vehicle (circles) for the indicated times. Cell surface receptor levels were detected by ELISA using CXCR7- or CXCR3-specific antibodies (11G8 and mAB160, respectively). Results were normalized to basal surface protein levels, and data represent the mean ± SEM of 4 experiments each performed in triplicate. (C) ELISA was performed as in B in cells pre-incubated for 2 h with the de novo protein synthesis inhibitor cycloheximide (10 µg/ml). (D) ELISA performed as in C on intact HEK293/CXCR7 cells treated with vehicle or 1 µM of bafilomycin A1 (Baf A1), 30 min prior to incubation with CXCL12. (E) C-terminal Ser/Thr clusters determine receptor fate after internalization. HEK293T cells were transiently transfected with CXCR7 wt (white bars) or with a chimeric receptor consisting of CXCR7 harboring the C-terminal sequence of CXCR3 (CXCR7-X3, filled bars). To assess the cell surface expression of the receptor, ELISA experiments were performed after 30 min or 3 hours of incubation with 10−8 M CXCL12. Data represent the mean ± SEM of 3 experiments each performed in triplicate. ***, p<0.001, **, p<0.01, and *, p<0.05 by one-way ANOVA and Bonferroni post test.
Figure 4.
The C-terminus of CXCR7 is constitutively ubiquitinated.
(A) CXCR7 gets deubiquitinated by CXCL12-stimulation. HEK293T cells were transfected as indicated and processed for immunoprecipitation of the HA-Ub (See Materials and Methods). (A) CXCR7 was stimulated with 10−8 M CXCL12 for 30 min, and removal of CXCL12 was performed by two washes of the cells and additional 30 min incubation with fresh chemokine-free media. Detection of the immunoprecipitated CXCR7 was done with the 11G8 antibody. HA-Ub expression was confirmed by blotting lysates using an anti-HA antibody and equal loading was controlled by detection of actin on the same blot. Molecular weight markers (kDa) are indicated on the right of the blot. (B) Detection of total CXCR7 protein expression by ELISA in the same cells.
Figure 5.
CXCR7/CXCR3 tail switch alters ubiquitination properties of the receptors.
(A) Immunoprecipitation experiments were performed in cells expressing chimeric receptors consisting on CXCR7 with CXCR3 C-terminus (CXCR7-X3) or the reciprocal CXCR3 with CXCR7 C-terminus (CXCR3-X7). Detection of the immunoprecipitated CXCR7 and CXCR3 was done with the 11G8 and mAB160 antibodies, respectively. HA-Ub expression was confirmed blotting lysates using an anti-HA antibody and equal loading was controlled by detection of actin on the same blot. Molecular weight markers (kDa) are indicated on the sides of the blots. (B) Detection of total CXCR7 protein expression by ELISA in the same cells.
Figure 6.
Real-time monitoring of receptor ubiquitination using BRET2.
HEK293T cells were transfected with Ub-GFP2 (white bars) or (G75A,G76A)-Ub-GFP2 (filled bars) and (A) CXCR7-RLuc, CXCR7 ΔC-RLuc, or CXCR7 ST/A-Rluc, (B) CXCR4-RLuc, or (C) CXCR3-RLuc. BRET2 was measured 30 min after stimulation with 10−8 M of CXCL12 (CXCL11 for CXCR3) by addition of coelenterazine 400a and immediate read out. Results are expressed in Net BRET normalized to basal as described in Materials and Methods. Data represent the mean ± SEM of 3 experiments each performed in triplicate. **, p<0.01, and ***, p<0.001, by Student t test.
Figure 7.
CXCR7 C-terminal Lys residues are important for correct trafficking of the receptor to the cell surface.
(A) [125I]CXCL12 radioligand binding in total membranes of HEK293T cells transfected with CXCR7 wt (filled circles) or CXCR7 K/A (open squares) shows that both receptors display the same affinity for CXCL12 (pKd = 9.7±0.1 and 9.5±0.1 respectively). (B) [125I]CXCL12 radioligand binding was performed in whole cells expressing pcDEF3 (mock) CXCR7 wt (WT) or CXCR7 K/A (K/A) (C) CXCR7 wt or CXCR7 K/A expressing cells were fixed (open bars) or fixed and permeabilized (filled bars) and CXCR7 was detected by ELISA using CXCR7-specific antibody 11G8. (D) Immunocytochemistry of HEK293T cells transfected with CXCR7 wt (upper panels) or CXCR7 K/A (lower panels). Cells were fixed (left panels) or fixed and permeabilized (right panels) and detection of CXCR7 was performed with 11G8 antibody. Scale bar represents 10 µm.
Figure 8.
Proposed model for regulation of CXCR7 trafficking.
CXCR7 requires ubiquitination of the Lys residues of its C-tail in order to reach the cell surface. Receptor activation by CXCL12 and subsequent phosphorylation of the C-terminal Ser/Thr residues results in β-arrestin recruitment by CXCR7 and receptor internalization in CCPs. In addition, β-arrestin scaffolds the interaction of CXCR7 with an unknown de-ubiquitinating enzyme (DUB) responsible for receptor deubiquitination. After chemokine degradation in early endosomes and due to the transient interaction of CXCR7 with β-arrestin, release of β-arrestin (and DUB) from the endocytosed receptor results in a CXCR7 able to undergo ubiquitination by a specific E3 ligase (E3) and subsequent delivery of the recycled receptor to the cell surface.